Moreover, RT-PCR confirmed that these cells expressed epithelial-related genes, such as cytokeratin 14, E-cadherin, and ΔNp63. Additionally, HERS cells showed low expression of CD44 and CD105 with absence of CD34 and amelogenin expressions. In conclusion, HERS cells have been successfully isolated using a selective digestion method, thus enabling future studies on the roles of these cells in the formation of cementum-like tissue in vitro.Hertwig’s epithelial root sheath (HERS) cells play a pivotal role during root formation of the tooth and are able to form cementum-like tissue. The aim of the present study was to establish a HERS cell line for molecular and biochemical studies using a selective digestion method. Selective digestion was performed by the application of trypsin-EDTA for 2 min, which led to the detachment of fibroblast-like-cells, with the rounded cells attached to the culture plate. The HERS cells displayed a typical cuboidal/squamous-shaped appearance. Characterization of the HERS cells using immunofluorescence staining and flow cytometry analysis showed that these cells expressed  Tooth development and periodontal tissue formation are regulated by a series of reciprocal interactions between the oral epithelium and mesenchymal cells. During tooth development, after crown formation, the apical mesenchyme continues to proliferate to form the developing periodontium while the inner and outer enamel epithelial cells fuse below the level of the cervical margin of the crown to develop a bi-layered epithelial sheath, which is called Hertwig’s epithelial root sheath (HERS) . After ret al. (2007) [Heterogeneous culture may cause variations in results due to differential expression in different cells. In a study by Sonoyama . 2007) , the per. (2007) –7, indic , the pe. (2007) –8 or pro. (2007) .in vitro studies have been performed to isolate and characterize HERS cells and certain derivative cell lines [Although several ll lines ,8,10,11,Periodontal ligaments were digested in a mixture of collagenase and dispase, and the single-cell suspensions were seeded in KGM. The primary cultures consisted of cells with a fibroblast-like appearance and rounded/squamous epithelial cell-like appearance . Therefoin vitro. The culture was positively stained using antibodies against pan-cytokeratin, E-cadherin, and p63. The same expression pattern was found in keratinocytes  [This study is the first report of the successful establishment of HERS cells using a new technique, selective digestion, to obtain pure HERS cells population from a primary heterogeneous cell culture after several attempts and optimizations. Based on the observation that epithelial cells attach more strongly to culture plates than fibroblast-like cells, the selective digestion method employs 0.25% trypsin-EDTA for 2 min. However, the prolonged exposure to trypsin can affect the integrity of the cell membrane, cell viability, and ultimately lead to cell necrosis ,23. Inde. (2010)  found thet al. (2007) [The selective digestion method was very efficient in promoting the detachment of the fibroblast-like cells in the initial heterogeneous culture, leaving the epithelial-like cells adhered to the culture plate. Thereafter, the HERS cells were cultured in KGM, which is specifically used to culture epithelial cells; hence, the few remaining fibroblast-like cells were removed from the culture by the subsequent sub-culturing processes. This result proves that KGM was specific to grow only epithelial cells ,8. Non-e. (2007)  reportedet al. (2011) [et al. (2007) [Under our conditions, the morphology of the HERS cells was that of typical cuboidal/cobblestone-shaped epithelial cells, which is in agreement with other studies ,8,10,11.. (2011) . In cont. (2007)  stated t. (2007) –27.et al. (2011) [in vitro [in vivo [Our immunofluorescence results showed that the HERS cells expressed the tested epithelial markers, whereas no staining was identified in the negative control, confirming the validity of our protocol, and that the staining was not caused by non-specific interactions of immunoglobulin molecules with the cells. Moreover, flow cytometry and RT-PCR results confirmed that the HERS cells positively expressed pan-cytokeratin, p63, and E-cadherin as epithelial markers and could be characterized as epithelial-like cells, consistent with other studies ,7,11. In. (2011) –33) exprin vitro  and in v[in vivo ,35 studiin vitro model for future studies.HERS cells are a unique epithelial population that plays a critical role during root formation, thus the establishment of a cell line is essential and would serve as an n = 40) were collected from adults, aged between 18 and 23 years old, who attended dental clinics of the School of Dental Sciences, Universiti Sains Malaysia, USM, Malaysia. The study was approved by the Human Research Ethics Committee of Universiti Sains Malaysia, USM, Malaysia. The teeth were delivered in Hank’s balanced salt solution  supplemented with 3% antibiotics/antimycotics . The periodontal ligament (PDL) tissues were gently separated from the root surfaces of the teeth using a fine forceps  (1:300), anti-E-cadherin  (1:50), and anti-p63  (1:100). For the antibodies against intracellular and nuclear proteins, a permeabilization solution (0.1% Triton X-100/PBS) was applied to the cells for 5 min. The primary antibodies were diluted in PBS + 10% goat serum and applied to the cells overnight at 4 °C. After washing with PBS three times for 5 min each, goat anti-mouse fluorescein (FITC)-conjugated secondary antibody 1 gG (1:200)  was applied, and the samples were incubated in the dark for 1 h at RT. Following this incubation, the cells were washed three times with PBS containing 0.1% Tween-20 and counterstained with 4′-6-diamidino-2-phenylindole (DAPI)  for 15 min; the cells were then washed with PBS. The slides were mounted in a fluorescent mounting medium (DAKO) before being viewed under a fluorescence image analyzer . The experiment was performed in triplicate. Human epidermal keratinocytes  were used as a positive control and isotype mouse IgG1as a negative control.HERS cells (at passage 3) were cultured in 4-well culture slides until they reached 70% confluence. After washing with phosphate-buffered saline (PBS) three times for 5 min each, the cells were then fixed with freshly prepared 4% paraformaldehyde (PAF) diluted with PBS for 10 min at room temperature (RT). The fixative solution was aspirated, and the cells were washed with PBS before being blocked with 10% normal goat serum for 1 h at RT. The following antibodies were used: monoclonal mouse anti-human Pan-cytokeratin  (0.2 μL); anti-E-cadherin  (20 μL); and anti-p63  (1 μL). Each primary antibody was incubated with 1 × 106 cells for 1 h in the dark at RT. After washing with PBS, goat anti-mouse fluorescein (FITC)-conjugated secondary antibody IgG (1:100) was applied for 30 min in the dark at RT. The cells were then resuspended in PBS solution and analyzed using flow cytometry . For the antibodies against intracellular and nuclear proteins, the cells were fixed with 1% paraformaldehyde for 10 min and permeabilized with ice-cold methanol for 10 min before incubation with the primary antibody. Keratinocytes were used as a positive control and isotype mouse IgG1 as a negative control. To further characterize HERS cells, mesenchymal markers were utilized following the same protocol: monoclonal mouse anti-human CD44  (1 μL); anti CD105  (10 μL); endothelial marker monoclonal mouse anti-human CD34  (10 μL).For the flow cytometry analysis, the HERS cells were harvested at passage 3 and washed with PBS. The following antibodies were used: monoclonal mouse anti-human Pan-cytokeratin  according to the manufacturer’s instructions. The obtained cDNA was used as the template for PCR. A housekeeping gene (GAPDH) was used to normalize all the reactions, and a no-template control (NTC) was included in all the reactions. Human-specific primers for the cytokeratin 14, E-cadherin, amelogenin, and GAPDH genes were designed according to the published cDNA sequences in GenBank, and the sequence for ΔNp63 as reported by Yang et al. 1998 [cytokeratin 14 and E-cadherin and 61.5 °C for p63, and 1 min of extension at 72 °C, followed by 1 step of a final extension at 72 °C for 5 min. The PCR products were separated on a 2% agarose gel and visualized using SYBR Green. The band intensities were then quantified using alpha digital software. The results were normalized to GAPDH, and compared as a ratio of each expressed gene. All the reactions were performed in triplicate.The HERS and keratinocytes were harvested at passage 3, and the total RNA was extracted using the RNeasy kit . Then, 1 μg of total RNA was reverse transcribed using SuperScriptal. 1998  . Thus, our data showed that HERS cells were successfully established, and their purity and identity were confirmed both morphologically and biochemically.


Thalassiosira pseudonana and Phaeodactylum tricornutum created an opportunity for extensive studies on the structure of these metabolic pathways. Based on sequence homology found in the analyzed diatomal genes, the biosynthesis of amino acids in diatoms seems to be similar to higher plants. However, one of the most striking differences between the pathways in plants and in diatomas is that the latter possess and utilize the urea cycle. It serves as an important anaplerotic pathway for carbon fixation into amino acids and other N-containing compounds, which are essential for diatom growth and contribute to their high productivity.Amino acids are not only building blocks for proteins but serve as precursors for the synthesis of many metabolites with multiple functions in growth and other biological processes of a living organism. The biosynthesis of amino acids is tightly connected with central carbon, nitrogen and sulfur metabolism. Recent publication of genome sequences for two diatoms  Bacillariophyceae) are a group of unicellular eukaryotic algae. Their characteristic trait is the highly ornamented siliceous cell wall called a frustule. It resembles a Petri dish since it is composed of two parts where one is slightly bigger than the other. This is also the origin of their name, as the term ‘diatom’ is derived from the Greek word diatomos meaning “cut in half”.Diatoms ; and the toxin-producing Pseudo-nitzschia multiseries , provide a valuable opportunity to explore genetic background for metabolic networks in diatoms. The sequencing data of the T. pseudonana and P. tricornutum were used to reconstruct biosynthetic pathways in diatoms and the results were presented on-line in the Kyoto Encyclopedia of Genes and Genomes [The recently sequenced genomes of four diatom species: The pelagic, centric eudonana ; the bencornutum ; the psy Genomes , and Dia Genomes . Neverthetc. The carbon backbones of amino acids are derived from central carbon metabolism: glycolysis , photosynthesis , oxidative pentose phosphate pathway , photorespiration , and TCA cycle  [Diatoms, like other photoautotrophs synthesize a whole range of amino acids for building proteins and other compounds such as polyamines , glutathacetate) .3 can be assimilated into glutamine and glutamate through the action of glutamine synthetase and glutamate dehydrogenase, respectively. In the genomes of both P. tricornutum and T. pseudonana two isoforms of glutamine synthetase (EC 6.3.1.2) can be found. The analysis of these four sequences with HECTAR software [T. pseudonana) and mitochondria (P. tricornutum). It is possible that in P. tricornutum glutamine synthetase I is dually localized in order to help reassmilate ammonia from the photorespiration. The mitochondrial localization of glutamine synthetase has been already reported in plants [P. tricornutum and T. pseudonana seems to be catalyzed by one enzymatic complex (EC 1.4.1.14 and 1.4.1.13). Moreover, ammonium can be used to form glutamate in reaction catalyzed by glutamate dehydrogenase (EC 1.4.1.4). Sequences of two isoforms of this enzyme were found in T. pseudonana and in P. tricornutum.Before inorganic nitrogen can be incorporated into amino acids it has to be reduced to ammonia. NHsoftware  suggestsn plants . The ammAspartate synthesis uses oxaloacetate as carbon backbone. The amino group is donated by the glutamate. This reaction is catalyzed by aspartate aminotransferase (EC 2.6.1.1) and in both sequenced diatoms two isoforms of this enzyme were found. In prokaryotes, aspartate can be used to create alanine through the action of aspartate decarboxylase (EC 4.1.1.12). However, no gene sequence assigned to this enzyme could be found. It seems that the synthesis of alanine is catalyzed primarily by alanine transaminases (EC 2.6.1.2) which transfer the amino moiety from glutamate onto pyruvate. Two isoforms have been found in the genomes of both diatom species.2 into ribulose-1, 5-bisphosphate by ribulose-1,5-bisphosphate carboxylase oxygenase (EC 4.1.1.39), which yields one 3-phosphoglyceric acid and one 2-phosphoglycolate in reaction of oxygenation. The latter product cannot be used in the Calvin-Benson cycle and is instead salvaged through the photorespiratory cycle. 2-phosphoglycolate after dephosphorylation and oxidation reactions gives rise to toxic glyoxylate, which in turn is the direct precursor for glycine synthesis  can be used in mitochondria to form carbamoyl phosphate, which is a substrate in the urea cycle. Both enzymes can be found in genomes of the two diatoms. The in plants the subsequent conversion of serine to hydroxypyruvate and its further reduction to glycerate allows resupplying of the Calvin-Benson cycle with 3-phosphoglycerate. One should note here, that Kroth et al. [P. tricornutum or T. pseudonana.In diatoms, as in plants, serine and glycine seem to be synthesized in photorespiratory glycolate pathway . The anaynthesis A. The fih et al.  could noChlamydomonas reinhardtii [Both diatoms can synthesize serine in a non-photorespiratory pathway B in a wanhardtii ,. This pT. pseudonana and P. tricornutum live in sulfate-rich environment, where the availability of sulfur for biosynthesis of cysteine and methionine is no limiting factor. Sulfate assimilation is a relatively expensive process, as it requires eight electrons to reduce sulfate to sulfide, which can be incorporated into cysteine [42− to S2− consumes 732 kJ mol−1. For comparison, the reduction of nitrate to NH3 requires also eight electrons and only 347 kJ mol−1 [Marine diatoms such as cysteine . The redkJ mol−1 .T. pseudonana, has been recently reviewed by Bromke and Hesse [T. pseudonana contains two sequences that could be annotated as ATP sulfurylase. One ORF encodes for the single enzyme and the second isoform encodes a composite enzyme consisting of ATP sulfurylase fused to adenosine-5′-phosphosulfate kinase (EC 2.7.1.25) and inorganic pyrophosphatase (EC 3.6.1.1). The first enzyme activates sulfate and produces adenosine-5′-phosphosulfate (APS), while the activity of latter the composite enzyme would result in synthesis of 3'-phospho-adenosine-5′-phosphosulfate (PAPS). The composite ATP sulfurylase is also found in P. tricornutum. The gene sequence analysis suggests the presence of a targeting sequence in the T. pseudonana composite ATP sulfurylase protein, which would direct the protein into plastids; hence, sulfate in plastids could be used to synthesize 3′-phospho-adenosine-5′-phosphosulfate [T. pseudonana. The analysis of protein sequence suggests that one of the APS reductases is localized in the plastid, while the other one is probably found in the cytosol [T. pseudonana contain a thioredoxin domain in their C- termini, in which a conserved motif CxxC can be found. The enzymes lack another pair of cysteines, which is a prerequisite for proper binding of the FeS cofactor [T. pseudonana protein extract reduces APS at very high rates [et al. [P. tricornutum and T. pseudonana only single copy of sulfite reductase could be identified.Sulfate reduction and the pathway of cysteine synthesis in diatoms , with spnd Hesse . Brieflyosulfate . Adenosiosulfate . Two seq cytosol . Both APcofactor , neverthgh rates ,31. Kopr [et al.  suggestsT. pseudonana, three genes encoding serine acetyltransferase and four O-acetylserine (thiol)lyase sequences were identified. The genome of P. tricornutum encodes one serine acetyltransferase and three O-acetylserine (thiol)lyase proteins.To form cysteine, sulfide is incorporated into O-acetylserine in a reaction catalyzed by the enzyme O-acetylserine (thiol)lyase (EC 2.5.1.47). The enzyme is regulated by serine acetyltransferase (EC 2.3.1.30), an enzyme that synthesizes O-acetylserine by acetylation of serine. Both enzymes, serine acetyltransferase and O-acetyserine(thiol)lyase, are necessary to build a functional cysteine synthase complex and their interaction enables an allosteric regulation of the cysteine formation . In the P. tricornutum and T. pseudonana. Moreover one sequence of aparaginase (EC 3.5.1.1) which catalyzes the hydrolysis of asparagine and the release of ammonia has been found in sequenced genomes both diatoms.Asparagine in diatoms can be synthesized from aspartate by either of two reactions, utilizing either glutamine or ammonia as the amino group donor . Both reT. pseudonana two enzymes are annotated as aspartate kinase. One of them has a C-terminal domain of homoserine dehydrogenase . The next step in this pathway is catalyzed by a NADPH-dependent aspartate semialdehyde dehydrogenase (EC 1.2.1.11), which forms aspartate 4-semialdehyde. The aspartate family pathway branches at this point and the carbon flux is distributed between the biosynthesis of lysine and the biosynthesis of methionine and threonine. In the following step of methionine and threonine synthesis, homoserine dehydrogenase (EC 1.1.1.3) reduces aspartate 4-semialdehyde yielding homoserine. This amino acid is not used for synthesis of proteins, but like serine its hydroxyl group can be derivatised by the transfer of an acetate or phosphate moiety. Homoserine synthesis in T. pseudonana can be catalyzed by one of the three homoserine dehydrogenases found in the genome as well as by the catalytic domain of one of the aspartate kinases. The last common reaction in the pathways of methionine and threonine synthesis is the phosphorylation of homoserine catalyzed by homoserine kinase (EC 2.7.1.39). Threonine then is synthesized by the catalytic action of threonine synthase (EC 4.2.3.1). Single isoforms of the latter two enzymes have been found in both diatoms.Threonine, together with methionine and lysine, belongs to the aspartate family of amino acids since their biosynthesis pathways start with aspartic acid . ThreoniT. pseudonana posesses a gene encoding an enzyme with the same catalytic domain and also similar to proteins from parasitic eukaryotes Leishmania sp. and Trypanosoma sp. [P. tricornutum has at least one sequence which could be annotated as cystathionine γ-synthase. In genomes of both diatoms, a sequence of homoserine acetyltransferase (EC 2.3.1.31) has been identified. It is not known if the acetylated homoserine is used in the synthesis of homocysteine in a reaction similar to the one found in fungi [de novo-synthesis of methionine. This last step is catalyzed by methionine synthase enzyme. There are two types of this enzyme present in diatoms. T. pseudonana utilizes only the efficient but cobalamin-dependent MetH type (EC 2.1.1.13), while P. tricornutum carries both MetH and a cofactor-independent MetE type (EC 2.1.1.14) of methionine synthase genes [P. tricornutum is similar to Chlamydomonas reinhardtii, which also has both types of methionine synthase [In the methionine synthesis pathway O-phosphohomoserine can be used also by a cytosolic enzyme cystathionine γ-synthase (EC 2.5.1.48) . Cystathsoma sp. . These osoma sp. , which sin fungi  or bactein fungi . Homocysse genes . In thissynthase .meso-2,6-diaminopimelate. In the final reaction of lysine biosynthesis, meso-2,6-diaminopimelate is used by diaminopimelate decarboxylase (EC 4.1.1.20). For all the enzymes named here a single copy of a respective gene was found in the genomes of P. tricornutum and T. pseudonana. The biochemical proof for a functional diaminopimelate pathway in pennate diatoms was delivered by Brown [Lysine biosynthesis shares the first two reactions with the pathways of methionine and threonine synthesis . The lasby Brown .P. tricornutum there are two ORFs thought to encode this enzyme and in the other diatom species only one copy has been found. 2,3-Dihydroxy-3-methylvaleric acid is a substrate for the activity of dihydroxy-acid dehydratase (EC 4.2.1.9) which produces the direct precursor of isoleucine 2-oxo-3-methylvalerate. Finally, a branched-chain amino acid transaminase (EC 2.6.1.42) catalyzes the transfer of an amino group from glutamate onto the oxoacid of isoleucine. In the genome of P. tricornutum five copies of branched-chain amino acid transaminases have been detected, while the genome of T. pseudonana encodes three isoforms.In plants, isoleucine is synthesized from threonine and it seems that this pathway functions in diatoms as well . The firThe pathway of valine biosynthesis is a four-step pathway that shares all of its steps with the parallel pathway of isoleucine biosynthesis . The syn+-dependent oxidation catalyzed by 3-isopropylmalate dehydrogenase (EC 1.1.1.85). This reaction is followed by a spontaneous decarboxylation producing an oxoacid of leucine, 4-methyl-2-oxopentanoate. The last reaction in this pathway is catalyzed by one of the branched-chain amino acid transaminases. If not mentioned differently, all enzymes in this pathway are present as a single copy gene in the genomes of the two sequenced diatoms.2-Oxoisovalerate, which is the direct substrate for valine is also a substrate in the pathway which yields leucine . Four ouenolpyruvate in reaction catalyzed by 2-dehydro-3-deoxyphosphoheptonate aldolase  . Each genome has at least three sequences annotated as 3-dehydroquinate synthase genes. The third enzyme of the chorismate biosynthesis pathway, 3-dehydroquinate dehydratase (EC 4.2.1.10) convert 3-dehydoquinate into 3-dehydroshikimate, which is reduced to shikimate in the fourth step by a shikimate-NADP oxidoreductase (EC 1.1.1.25). A subsequent phosphorylation of shikimate to form shikimate-3-phosphate is catalyzed by shikimate kinase (EC 2.7.1.71). The genomes of both diatoms have a single copy of 3-dehydroquinate dehydratase and shikimate:NADP oxidoreductase. The fifth reaction in the pathway is catalyzed by an enzyme for which two copies were found in the genomes of each T. pseudonana and P. tricornutum. Sikimate 3-phosphate and phosphoenolpyruvate are used to produce 5-enolpyruvylshikimate-3-phosphate and orthophosphate. This reaction is catalyzed by 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19). Two genes for this enzyme were found in both diatomal genomes. The final step in the synthesis of chorismate, the dephosphorylation of 5-enolpyruvylshikimate-3-phosphate to form chorismate, is catalyzed by chorismate synthase (EC 4.2.3.5), which is encoded by only one gene in each of the diatoms.In higher plants and in Chlamydomonas three aromatic amino acids: phenylalanine, tyrosine and tryptophan, are derived from a common precursor, chorismate . Basing .5.1.54) . Two genP. tricornutum and one in T. pseudonana genome. On basis of the identified genes it is likely that the pathway of phenylalanine and tyrosine goes via phenylpyruvate and not p-hydroxyphenylpyruvate. The other possibility is a branching to the final products after transamination of the intermediate arogenate. A gene for prephenate dehydrogenase (EC 4.2.1.51) has been found in P. tricornutum, which suggest the synthesis of phenolpyruvate in the diatomal cell. This compound is used by one of four phyenylalanine:2-oxoglutarate transaminases (EC 2.6.1.57) found in the P. tricornutum genome. Four genes of the transaminase were also found in T. pseudonana. In both genomes, the genes annotated as prephenate:aspartate and prehenate:glutamate transaminases were found. This fact does not exclude the functioning of an alternative synthesis pathway in which the final phenylalanine synthesis reaction is the transamination. In each diatomal genome only a single gene was found which could be annotated as a phenylalanine 4-monooxygenase (EC 1.14.16.1). Since no genes of enzymes catalyzing the synthesis of tyrosine from arogenate were found, this suggests that the oxygenation of phenylalanine is the only way of achieving tyrosine biosynthesis.Prephenate is the next intermediate in the synthesis pathway of tyrosine and phenylalanine. It is produced from chorismate by the action of chorismate mutase (EC 5.4.99.5) for which two genes were found in enolpyruvyl side chain by a transfer of the amino group from glutamine (P. tricornutum two sequences could be annotated as the genes coding this enzyme. 5′-phosphoribosylanthranilate undergoes isomerisation by the action of the phosphoribosylanthranilate isomerase (EC 5.3.1.24). One sequence of this enzyme was found in the P. tricornutum genome. Following reaction of indole-3-glycerol-phosphate synthase (EC 4.1.1.48) produces indole-3-glycerolphosphate from 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose-5-phosphate. There are two ORF’s for this enzyme in the genome of P. tricornutum. In plants, the two last steps in this pathway are catalyzed by a single enzyme, tryptophan synthase (EC 4.2.1.20), which is composed of two α and two β subunits [P. tricornutum there is one gene for each of the two subunits of the tryptophan synthase complex. In contrast, T. pseudonana has only one ORF in which two catalytic subdomains TrpA and TrpB can be found. This is another example of a fused enzyme catalyzing subsequent reactions in this diatom. Despite the missing data on the genes of this pathway in T. pseudonana, one can assume that the general architecture of the pathway is as it is described for P. tricornutum, with the exceptions mentioned here.The biosynthesis of tryptophan in diatoms seems to be similar to the one found in plants, where the pathway begins with the conversion of chorismate to anthranilate by the elimination of the lutamine . As in osubunits . Each ofP. tricornutum and all but one in case of T. pseudonana. The histidine synthesis begins with the ATP-dependent pyrophosphorylation of ribose-5-phosphate catalyzed by ribose-phosphate diphosphokinase (EC 2.7.6.1) to form 5-phosphoribosyl diphosphate (P. tricornutum and T. pseudonana genomes. PhosphoribosylformiminoAICAR-P further is converted by a phosphoribosylformiminoAICAR-P isomerase (EC 5.3.1.16) to phosphoribulosylformiminoAICAR-P. The following step, a conversion of phosphoribulosylformiminoAICAR-P to erythro-imidazoleglycerol phosphate and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), requires two catalytic functions, a transamidation (with glutamine as amide donor) and a cyclization reaction, both of which are catalyzed in diatoms by the single enzyme imidazole glycerol phosphate synthase (2.4.2.-). It seems that diatoms, like plants, use the single enzyme, whereas prokaryotes use two separate enzymes to catalyze this reaction [The pathway of histidine biosynthesis seems to proceed in similar way to that found in plants . All genhosphate . In the reaction . In the reaction . The twoP. tricornutum. For the rest of the reactions described here, only one copy of a gene encoding respective enzyme has been found. In the genome of T. pseudonana each enzyme is represented by one gene and so far no histidinol phosphate phosphatases have been annotated.The dehydratation of erythro-imidazoleglycerol phosphate and the dephosphorylation of histidinol-phosphate can be catalyzed by two enzymes, as for each them two ORFs have been found in the genome of P. tricornutum three sequences were annotated as pyrroline-5-carboxylate reductase, whereas only single gene was found in T. pseudonana. Alternatively, proline can be synthesized from ornithine, which is first transaminated by ornithine-delta-transaminase (EC 2.6.1.13) producing glutamate-semialdehyde. Both diatoms have a single gene for this enzyme. Further reactions are the same as in the “glutamate-derived” pathway described above.Glutamate is the common precursor for the synthesis of both amino acids. In addition, ornithine, an important intermediate in the arginine biosynthetic pathway, can serve as an alternative precursor for proline biosynthesis. On the basis of gene homology there seem to be two plant-like pathways for proline biosynthesis . The maiN-acetylglutamate synthase (EC 2.3.1.1). There are two genes for N-acetylglutamate synthase in the genome of T. pseudonana and one in the genome of P. tricornutum. The second step of the ornithine pathway is the phosphorylation of N -acetylglutamate by N-acetylglutamate kinase (EC 2.7.2.8). N-acetylglutamyl-phosphate then undergoes a NADPH-dependent, dephosphorylating reduction catalyzed by N-acetylglutamyl reductase (EC 1.2.1.38) to produce N-acetylglutamyl semialdehyde. The product of this reaction is further converted into N-acetylortnithine by acetylornithine transaminase (EC 2.6.1.11) with glutamate as the amino group donor. A subsequent reaction catalyzed by action of amidoacylase (EC 3.5.1.14) produces ornithine and acetate. Sequences of ornithine transacetylase (EC 2.3.1.35) have not been found in the genome of T. pseudonana or in P. tricornutum. This is one of the differences in metabolic pathways between diatoms and plants, where this enzyme catalyzes the synthesis of ornithine. The conversion of ornithine to arginine proceeds in three steps of the urea cycle. The genes of the urea cycle were first described in T. pseudonana by Armbrust et al. [P. tricornutum by Bowler et al. [et al. [2 is used by mitochondrial carbamoyl-phosphate synthases to form a substrate for the urea cycle – carbamoyl-phosphate. Ornithine is converted to citrulline by reaction with carbamoyl-phosphate catalyzed by ornithine carbamoyltransferase (EC 2.1.3.3). In a subsequent reaction citrulline reacts with aspartate in an ATP-dependent reaction catalyzed by argininosuccinate synthase (EC 6.3.4.5). The product of this reaction, argininosuccinate is split by argininosuccinate lyase (EC 4.3.2.1) to arginine and fumarate. In the last step of the urea cycle, arginine is cleaved by arginase (EC 3.5.3.1.) giving urea and citrulline.Arginine biosynthesis in diatoms is tightly connected with urea cycle in which ornithine is converted to amino acids. In diatoms and in plants ornithine synthesis is initiated by the acetylation of glutamate using acetyl-CoA  Figure . The reaEC 3.5.1. producesr et al. . The fun [et al. . Diatoms [et al. . The ammThalassiosira pseudonana and Phaeodactylum tricornutum are valuable resources in the exploration of metabolic pathways in these ecologically important organisms. Despite their complex genetic background of diatoms, which involves at least two events of endosymbiosis and horizontal gene transfer, the general picture of amino acid biosynthesis pathways looks very similar to the one observed in higher plants or green algae. However, there are differences which make diatoms remarkable. One of them is the fully functional urea cycle, which greatly influences nitrogen metabolism and the synthesis of amino acids. Another interesting feature observed in the genomes of the discussed diatoms is the fact that enzymes catalyzing subsequent steps can be fused into one ORF like it is observed for ATP sulfurylase and tryptophan synthase. The genome sequence data are almost complete but there are still open questions concerning the details of the amino acids biosynthesis pathways. A thorough mining through the available sequence data will probably reveal more interesting examples of pathways, gene fusions, transfers and other mutations that helped diatoms to become such an abundant and successful group of organisms. Nevertheless, a pathway analysis based on homologies does not allow the understanding of the regulatory and adaptive mechanisms behind the network of reactions. These pathway predictions as well as questions of regulatory mechanisms of metabolism in diatoms will be answered with the use of molecular genetics methods.The sequenced genomes of diatoms


Despite the widespread use of interferon-γ release assays (IGRAs), their role in diagnosing tuberculosis and targeting preventive therapy in HIV-infected patients remains unclear. We conducted a comprehensive systematic review to contribute to the evidence-based practice in HIV-infected people.+ T-cell count <200 (11.6% for QFT-GIT and 11.4% for T-SPOT.TB) than in those with CD4+ T-cell count ≥200 (3.1% for QFT-GIT and 7.9% for T-SPOT.TB).We searched MEDLINE, Cochrane, and Biomedicine databases to identify articles published between January 2005 and July 2011 that assessed QuantiFERON®-TB Gold In-Tube (QFT-GIT) and T-SPOT®.TB (T-SPOT.TB) in HIV-infected adults. We assessed their accuracy for the diagnosis of tuberculosis and incident active tuberculosis, and the proportion of indeterminate results. The search identified 38 evaluable studies covering a total of 6514 HIV-infected participants. The pooled sensitivity and specificity for tuberculosis were 61% and 72% for QFT-GIT, and 65% and 70% for T-SPOT.TB. The cumulative incidence of subsequent active tuberculosis was 8.3% for QFT-GIT and 10% for T-SPOT.TB in patients tested positive (one study each), and 0% for QFT-GIT (two studies) and T-SPOT.TB (one study) respectively in those tested negative. Pooled indeterminate rates were 8.2% for QFT-GIT and 5.9% for T-SPOT.TB. Rates were higher in high burden settings (12.0% for QFT-GIT and 7.7% for T-SPOT.TB) than in low-intermediate burden settings (3.9% for QFT-GIT and 4.3% for T-SPOT.TB). They were also higher in patients with CD4IGRAs have suboptimal accuracy for confirming or ruling out active tuberculosis disease in HIV-infected adults. While their predictive value for incident active tuberculosis is modest, a negative QFT-GIT implies a very low short- to medium-term risk. Identifying the factors associated with indeterminate results will help to optimize the use of IGRAs in clinical practice, particularly in resource-limited countries with a high prevalence of HIV-coinfection. Tuberculosis is one of the leading causes of mortality in people living with human immunodeficiency virus (HIV) worldwide, particularly in sub-Saharan Africa, where it is responsible for up to half of HIV-related deaths HIV co-infection increases the risk of tuberculosis either by facilitating reactivation of a remote latent infection (LTBI) or by favoring the progression of a recently acquired infection towards active disease. Therefore, rapid identification and early treatment of active tuberculosis cases in order to interrupt further transmission, as well as the detection and treatment of LTBI to prevent progression to active disease, are crucial for controlling HIV-associated tuberculosis M. tuberculosis-specific antigens which are not present in most non-tuberculous mycobacteria or in the bacillus Calmette-Guérin vaccine T-cell-based interferon-gamma (IFN-γ) release assays (IGRAs) constitute a promising alternative to TST for diagnosing tuberculosis infection. IGRAs use highly In low-burden tuberculosis settings, IGRAs have shown better specificity and equal or greater sensitivity than TST for the detection of tuberculosis infection, and a better correlation with the intensity of exposure to a source of infection Although IGRAs were not developed to replace conventional microbiological methods for the diagnosis of active tuberculosis disease, they may have an adjunctive role in symptomatic patients with suspicion of active disease by complementing clinical-radiographic and epidemiological data to guide diagnosis work-up. Therefore, knowing how HIV infection compromises the IGRAs' ability to detect tuberculosis infection in patients with active disease is essential in order to determine their role in different clinical and epidemiological settings.+ T-cell depletion affects IGRA performance, and tried to identify differences according to tuberculosis burden settings and HIV infection status.We conducted a comprehensive systematic review (SR) to assess the sensitivity and specificity of IGRAs for the diagnosis of active tuberculosis disease, their value to predict development of subsequent active tuberculosis, and the proportion of indeterminate results in HIV-infected adults. Whenever feasible, we assessed how HIV-associated CD4This SR was conducted in accordance with the PRISMA statement We systematically searched for studies published between 1 January 2005 and 31 July 2011 that evaluated the diagnostic performance of IGRAs for tuberculosis or LTBI in HIV-positive adult populations . We searched MEDLINE, the Cochrane Central Register of Controlled Trials, and the Biomedicine Database (IME) of the Spanish National Research Council (CSIC). Searches comprised a combination of the following terms: “HIV”, “immunosuppressed patients”, “tuberculosis”, “latent tuberculosis infection”, “QuantiFERON”, “QuantiFERON-TB Gold”, “T-SPOT.TB”, “interferon-gamma release assays”, and “T-cell assays”, as listed in titles, abstracts or text words. Searches were limited to studies published in English or Spanish. We also reviewed citations of the original and review articles, and guidelines for additional references. When necessary, we contacted the authors of the studies for additional information.For our analysis, we selected only prospective studies that usedthe commercial tests QuantiFERON®-TB Gold In-Tube and T-SPOT®.TB performed in blood with 16–24 h of incubation. We excluded studies of non-commercial IGRAs or studies based on the old version of the ELISA assay (QuantiFERON®-TB Gold), as well as studies presenting non-original data, conference abstracts, editorials, reviews, guidelines, and studies conducted in animals.We checked the quality of the studies used to calculate assay accuracy with the QUADAS check list In the case of indeterminate results, we appraised the quality of the studies by assessing whether or not a definition was given in the To evaluate the quality of the studies that assessed the risk of subsequent tuberculosis according to the result of an IGRA assay, we used the Newcastle-Ottawa Scale (NOS) for non-randomized cohort studies + cell count, development of active tuberculosis, TST results, indeterminate test results  and fraction of individuals with true positive, false negative, true negative and false positive results for the calculation of the test sensitivity and specificity.Two researchers (M.S. and L.M.) independently compiled the data using a standardized data extraction sheet. Discrepancies were resolved by discussion and consensus. The following data were extracted: year of publication, period and country, number of participants, gender, test evaluated, CD4M. tuberculosis, a positive nucleic acid amplification test, or characteristic histopathological findings and response to specific treatment) that was still untreated or had been treated for less than two weeks. Indeterminate results were included as false negatives. For the specificity calculation, we selected studies that had enrolled patients with suspected active tuberculosis . Results due to low IFN-γ production in the phytohaemagglutinin (PHA)-stimulated well or high background IFN-γ production were defined as indeterminate. Since T-SPOT.TB tests not performed due to insufficient PBMC cells are usually excluded from the analyses, we used the term “failure tests” for unrealizable tests. We assessed the effect of immunosuppression on sensitivity and indeterminate result rates by pooling and stratifying the results for 200 CD4+ T-cell count threshold.We assessed the following outcomes for each study and pooled them when feasible: sensitivity and specificity for active tuberculosis, predictive value for incident active tuberculosis, and rates of indeterminate results. The following definitions were used: sensitivity refers to the proportion of culture-proven tuberculosis patients who had a positive IGRA test, and specificity refers to the proportion of symptomatic non-tuberculosis patients who had a negative IGRA test. For the sensitivity calculation, we included only patients with confirmed tuberculosis , low- to intermediate-burden  I2 statistic. Forest plots were constructed to show the effect size of all the studies and the variability of the pooled estimates. The analyses were performed with MetaAnalyst software We calculated combined estimates of pooled sensitivity, specificity and the 95% confidence interval (CI). The pooled effect for binary outcomes was presented as the difference with the 95% CI. A random-effects synthesis model meta-analysis was used to pool the effect across the studies. Inconsistency was quantified by the Of the total of 677 citations identified, 38 were eligible for analysis  [19]–[56Eight of the 20 studies (40%) used to calculate sensitivity and specificity met all the quality indicators, ten (50%) met between 75% and 100%, and two (10%) met less than 75%.Indeterminate results due to high production of IFN-γ in the negative control were either not defined as such or excluded from the analysis in 32% of studies with QFT-GIT and in 28% with T-SPOT.TB. The results for the two types of indeterminate results were reported separately in 27% of studies with QFT-GIT and in 32% with T-SPOT.TB. Only three studies (16%) provided data on the T-SPOT.TB tests not performed because of insufficient quantities of cells.As for the studies evaluating the ability of IGRAs to predict subsequent tuberculosis, follow-up was adequate in all three , and the exposed sample was representative of the HIV population; however, there was no adequate outcome assessment (pre-test and during follow-up) in any of them, and the number of incident tuberculosis cases was low . More detailed information on the quality of the studies is available upon request.2I = 46.6%) for QFT-GIT and 65%  for T-SPOT.TB  for QFT-GIT and 79%  for T-SPOT.TB. The results of simultaneous TST and QFT-GIT in patients with active tuberculosis were reported in five studies I2 = 0.0%) for QFT-GIT and 60%  for TST. T-SPOT.TB was compared with TST in one study with 13 patients Five studies compared the sensitivity of QFT-GIT and T-SPOT.TB head to head I2 = 9.8%) and 79%  in HIV-infected and HIV-uninfected patients respectively. As for T-SPOT.TB, three studies compared sensitivity between HIV-infected and uninfected patients (55 HIV-infected and 364 HIV-uninfected) I2 = 39.1%) in HIV-infected and 90%  in HIV-uninfected patients. Six studies, covering 634 participants with tuberculosis (113 HIV-infected and 521 HIV-uninfected) evaluated the sensitivity of QFT-GIT according to HIV status + cell counts on sensitivity was evaluated in three studies with QFT-GIT + cells + T-cell counts between patients with positive and negative results + cells  than in those with ≥200 CD4+ cells  + T-cell counts + cells <200  than in those with CD4+ cells ≥200 cells  The effect of CD42I = 93.2%) for QFT-GIT, and 70%  for T-SPOT.TB   . When thI2 = 47.0%) (I2 = 48.3%). Of the 153 indeterminate results derived from these three studies, 51 (33.3%) were due to failure to perform the test, 50 (32.7%) were due to low IFN-γ production in the PHA-stimulated well, and 52 (34%) to high background IFN-γ production.Eighteen studies including a total of 2236 participants reported data on indeterminate results with T-SPOT.TB = 47.0%) . When thI2 = 46.9%) for QFT-GIT, and 6.1%  for T-SPOT.TB (In eight studies that compared QFT-GIT and T-SPOT.TB head to head -SPOT.TB .I2 = 44.5%) for QFT-GIT and 7.7%  for T-SPOT.TB. Pooled indeterminate rates in low/intermediate-burden countries were 6.4%  for QFT-GIT and 3.5%  for T-SPOT.TB. When stratified for type of patients , pooled indeterminate rates for QFT-GIT were higher for patients with active tuberculosis  and for symptomatic patients  than for those screened for LTBI . Pooled indeterminate rates for T-SPOT.TB were higher for symptomatic patients  than for those screened for LTBI .The pooled indeterminate rates for high-burden countries were 12.0% , as was the case of T-SPOT.TB   .I2 = 34.7%) for CD4+<200, and 3.1%  for CD4+ ≥200  for QFT-GIT, and 11.4%  for CD4+<200, and 7.9%  for CD4+≥200  for T-SPOT.TB , type of evaluated people , and setting (high-burden and resource-limited vs. low-burden and high-income settings), but are also due to differences in the criteria used for reporting data. Indeterminate results due to high-background IFN-γ production, as well as failure T-SPOT.TB tests due to an insufficient number of PBMCs, are often not counted as such and are excluded from the analyses. Therefore, the calculation of indeterminate result rates and their association with potentially influencing factors will inevitably be compromised by these limitations. Interestingly, the types of indeterminate results were not equally distributed for the two assays. While low IFN-γ production upon stimulation with PHA (positive control) accounted for more than 90% of the indeterminate results with the QFT-GIT assay, half of the indeterminate T-SPOT.TB assays were due to high-background IFN-γ production (negative control).Indeterminate results, due either to low IFN-γ production in the positive control or to high IFN-γ production in the negative control, may negatively affect the overall utility of IGRAs. The proportion of indeterminate results in the current SR showed huge differences across studies, ranging from no indeterminate results at all to rates as high as 25% and 33% for QFT-GIT and T-SPOT.TB respectively + T-lymphocyte depletion either as a cause or as a consequence of the disease, which may cause anergy and indeterminate IGRA results The pooled indeterminate rates for the two assays were higher in high-burden settings than in low-burden settings. They were also higher in patients with symptoms suggestive of tuberculosis or culture-confirmed tuberculosis than in those screened for LTBI. Because studies that enrolled patients with active tuberculosis were mainly carried out in high-burden countries, whilst those that enrolled patients screened for LTBI were from low-burden countries, further analyses to determine which of the two factors has a greater influence on the occurrence of indeterminate results cannot be performed. On the one hand, HIV-infected patients usually have profound CD4Our SR has limitations. First, the validity of the results is limited by the inconsistency across the studies. This heterogeneity persisted after performing subgroup analyses. Second, the main body of literature on active tuberculosis comes from high-burden tuberculosis and resource-limited settings, which limits the generalization of our estimates. Conversely, studies for the prediction of subsequent development of active disease in HIV-infected patients were exclusively from low-burden countries. Therefore, the low risk of subsequent active tuberculosis for patients testing negative on QFT-GIT obtained in two low-burden countries in Europe cannot be extrapolated to countries with high burdens of tuberculosis such as Sub-Saharan African countries. Finally, the lack of an adequate standard for latent tuberculosis infection is a inherent limitation to every SR to draw confident estimates on the capacity of IGRA tests to detect tuberculosis infection in people without evidence of active disease.Nonetheless, some relevant conclusions may be drawn from this SR. First, the current evidence indicates that neither IGRA is able to replace conventional microbiological diagnosis of tuberculosis in HIV-infected patients. Second, QFT-GIT, if the low risk of subsequent active tuberculosis in HIV-infected patients testing negative is confirmed, could replace TST for targeting at-risk patients for chemoprophylaxis in low-burden tuberculosis countries. Third, potential causes of invalid tests, such as delayed incubation and other operational factors, should be addressed in order to improve the performance of IGRAs, particularly in resource-limited high-burden tuberculosis countries with high HIV-coinfection prevalence.


An increased risk of renal cell carcinoma (RCC) has been linked with obesity and metabolic syndrome. However, the mechanisms by which lipid metabolic disorders affect the development of RCC remain unclear and highly controversial. Integrin-linked kinase (ILK) is a serine/threonine protein kinase involved in the regulation of tumor cell growth and angiogenesis. In the present study, the effect of free fatty acids in the promotion of RCC progression was investigated by upregulating ILK. Results of the MTT assay indicated that treatment of 786-O cells with oleic acid induced a concentration-dependent increase in cell viability. Flow cytometry analysis revealed that the effect of oleic acid on cell apoptosis was not significant. Following treatment with oleic acid, the expression of ILK, phospho-Akt and G protein-coupled receptor 40 (GPR40) was increased in 786-O cells. These effects were reversed when the expression of ILK was downregulated using specific small interfering RNA. These results indicate that free fatty acids are associated with the development of renal cell carcinoma via activation of the GPR40/ILK/Akt pathway, revealing a novel mechanism for the correlation between metabolic disturbance and renal carcinoma. Renal cell carcinoma (RCC), the most common pathological pattern, accounts for 3% of adult malignancy tumors and worldwide incidence continues to increase steadily each year . The 5-yin vitro and the mechanism by which FFAs function was determined.FFAs have been revealed to stimulate intracellular signal transduction, functioning as second messengers in vascular smooth muscle and tumor cells ,10; howeOleic acid and de-fatty bovine serum albumin (d-BSA) were purchased from Sigma-Aldrich . Oleic acid was supplemented with d-BSA, which functioned as a carrier to ensure sufficient dissolution (mol/mol <2). The annexin V-FITC apoptosis detection kit was purchased from Biosea Biotechnology Co. Ltd. . ILK small interfering RNA (siRNA) and control non-silencing siRNA were purchased from Cell Signaling Technology Inc. . Polyclonal anti-ILK, anti-Akt, anti-p-Akt ser473 and anti-G protein-coupled receptor 40 (GPR40) antibodies were purchased from Cell Signaling Technology.Human RCC cell line, 786-O, was obtained from American Type Culture Collection  and routinely cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin and streptomycin. For treatment, cells were cultured in growth medium for 24 h and then the medium was replaced with oleic acid-enriched medium at various concentrations of oleic acid . The control group received d-BSA alone at an equal concentration.The study was approved by the Ethics Committee of Peking University People’s Hospital, Beijing, China.3 cells/well) were seeded in 96-well microtitre plates and incubated for 24 h in 100 μl culture medium. Cells in the experimental group were then treated with 0.05, 0.1 or 0.2 mmol/l oleic acid for 48 h. MTT [20 μl (5 g/l)] was added to the cells which were then cultivated for a further 4 h. Following the removal of the supernatant fluid, 150 μl/well DMSO was added to the cells which were agitated for 15 min. Absorbance was measured at 490 nm by an ELISA reader. Untreated 786-O cells served as controls. Each assay was repeated three times. The relative growth rate of 786-O cells was calculated using the following equation: cell viability rate (%) = (ODoleic acid/ODcontrol) × 100.Cells  was mixed with 5 μl annexin V-FITC and 5 μl PI for 15 min, then 400 μl 1X binding buffer was added. Analysis was performed using a FACStar cytofluorometer with CellQuest Pro software.The extent of apoptosis was evaluated by annexin V-FITC and flow cytometry. Cells were grown at a density of 1×10siRNA duplexes specific for human ILK were used to specifically knock down ILK. For the transfection procedure, cells were grown to 60% confluence and ILK and control siRNA were transfected using the Oligofectamine reagent according to the manufacturer’s instructions. Briefly, Oligofectamine reagent was incubated with serum-free medium for 10 min. Subsequently, a mixture of respective siRNA was added. Following incubation for 15 min at room temperature, the mixture was diluted with medium and added to each well. The final concentration of siRNA in each well was 100 nmol/l. Following culture for 32 h, cells were washed and resuspended in oleic acid-enriched culture medium for an additional 48 h for cell growth assays and western blot analysis.Cell lysates were boiled with 5X loading buffer and then fractionated by SDS-PAGE. Proteins were transferred to PVDF membrane and incubated with primary specific antibodies against GPR40, ILK, Akt and phosphor-Akt in 5% milk. Blots were washed and horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies were applied. Blots were washed, transferred to freshly made enhanced chemiluminescence solution for 5 min and images were captured using the ImageQuant Gel imaging system.Data are expressed as mean ± SD for groups and analyzed using the Student’s t-test to determine differences between the oleic acid-treated and control groups. P<0.05 was considered to indicate a statistically significant difference.To examine the function of oleic acid on the human RCC cell line, 786-O cells were cultured and treated with various concentrations of oleic acid and cell viability was detected using the MTT assay. Percentages of viable cells when cultured with oleic acid  relative to the control-treated cells were determined at 4 h. The results indicate that oleic acid treatment induced a marked increase in the proliferation of 786-O cells in a dose-dependent manner .To determine the effect of oleic acid on the apoptosis of 786-O cells, annexin V/PI was used. It was observed that, following treatment of 786-O cells for 48 h with oleic acid , the total percentage of apoptotic cells was not directly associated with oleic acid concentration, decreasing from 2.42 to 2.33 and 2.25%, compared with the control cells (2.04%) (0.05, 0.1 and 0.2 mmol/l oleic acid, respectively; Since oleic acid treatment induced 786-O cell proliferation and delayed cell apoptosis, the effect of oleic acid on specific cell proliferation regulatory molecules was analyzed. Western blot analysis revealed that oleic acid treatment induced upregulation of ILK and phospho-Akt protein, a key regulator of cell proliferation, in a dose-dependent manner . To deteTo determine the role of ILK on 786-O cell proliferation, siRNA tranfection was performed to downregulate ILK expression. Cell viability was suppressed following transfection with ILK siRNA and treatment with oleic acid  and the Previous epidemiological and animal studies have identified an association between dietary fatty acids and metabolic diseases characterized by hyperlipidemia and an elevation in circulating FFAs and have indicated that this association also correlates with enhanced cancer risk –16. The Oleic acid is an n-9 monounsaturated fatty acid, which activates G protein-coupled receptors and phosphorylates ERK1/2 to induce cancer cell proliferation in breast cancer . The conet al found that GPR40 was highly expressed in ob/ob mice and may be involved in cell proliferation (GPR40 is a membrane-bound receptor paired with medium- and long-chain fatty acids as endogenous ligands . Briscoeferation . In the feration ,25. In tferation . To dateferation . ConsistIn summary, the results of this study indicate the following cascade of events in response to oleic acid in 786-O cells . Unsatur


C. elegans. We found that in this tissue, animals display a tubular mitochondrial network, which fragments with increasing age. This fragmentation is accompanied by a decrease in mitochondrial volume. Mitochondrial fragmentation and volume loss occur faster under conditions that shorten lifespan and occur slower under conditions that increase lifespan. However, neither mitochondrial morphology nor mitochondrial volume of five- and seven-day old wild-type animals can be used to predict individual lifespan. Our results indicate that while mitochondria in body wall muscles undergo age-dependent fragmentation and a loss in volume, these changes are not the cause of aging but rather a consequence of the aging process.Mitochondrial dysfunction is a hallmark of skeletal muscle degeneration during aging. One mechanism through which mitochondrial dysfunction can be caused is through changes in mitochondrial morphology. To determine the role of mitochondrial morphology changes in age-dependent mitochondrial dysfunction, we studied mitochondrial morphology in body wall muscles of the nematode  Caenorhabditis elegans, for instance, have revealed that sarcopenia is also a feature of aging in nematodes [C. elegans is a suitable model to address diverse aspects of sarcopenia [The age-associated degeneration of skeletal muscle function termed ‘sarcopenia’ leads to debilitating conditions among the elderly . Low skeematodes . Due to rcopenia .C. elegans have revealed that lifespan is influenced by gene activity, the environment, certain chemicals as well as stochastic factors [C. elegans shows a linear inverse-correlation to temperature of growth (for temperatures between 16°C to 25.5°C) [C. elegans IGF-1 receptor tyrosine kinase DAF-2  induces the activation of the PI3 kinase AGE-1 , which leads to the phosphorylation of the FOXO transcription factor DAF-16. When phosphorylated, DAF-16 is retained in the cytoplasm and therefore unable to modulate gene expression [daf-2 or age-1, lead to the nuclear localization of DAF-16 and an increased lifespan, while loss-of-function mutations in daf-16 lead to a decreased lifespan [daf-16 and - apart from affecting lifespan - also cause slow growth and slow motility as well as a decrease in brood size [clk-1 , whose gene product is required for ubiquinone biosynthesis, or loss-of-function mutations in the gene isp-1 , which encodes a Rieske iron-sulfur protein of the cytochrome bc1 complex (complex III), cause longevity [Studies in  factors , 10. For 25.5°C) . Further 25.5°C) . The actpression . In resppression . Hence, lifespan , 12-16. lifespan , 17. Theood size . For exaongevity .Although numerous processes have been implicated in age-associated muscle degeneration , 20, mitC. elegans [in vitro study using muscle cells derived from rat suggested that mitochondria are more fragmented in aging muscle cells [Drp1 (Dynamin-related protein 1), which encodes a dynamin-related GTPase that promotes mitochondrial fission, was observed in the in vitro study using rat muscle cells [The role of changes in mitochondrial morphology in age-dependent mitochondrial dysfunction remains poorly understood. Electron microscopic analysis of biopsies have revealed interconnected mitochondrial networks with giant mitochondria that have ultrastructural abnormalities in skeletal muscle cells of older humans . Further elegans . In contle cells . The mecle cells , anotherle cells . Furtherle cells . Howeverle cells . This suC. elegans, similar to what has been observed in human muscle biopsies, a global decrease in mitochondrial activity occurs with age. Specifically, the activity of NADH:ubiquinone oxidoreductase (complex I) decreases, as does oxygen consumption. Furthermore, with age, carbonylated proteins accumulate in mitochondria [C. elegans.In chondria . Using echondria . To analmyo-3 promoter (Pmyo-3mitoGFP), which is active in body wall muscle cells [C. elegans.To monitor mitochondrial morphology in body-wall muscle cells with age, we used a strain expressing mitochondrial matrix targeted GFP (mitoGFP) under the control of the le cells . Althougle cells -34, we omyo-3mitoGFP) and the reagent used to immobilize animals for imaging (levamisole), we confirmed these age-dependent changes in mitochondrial morphology and volume using a different mitochondrial reporter and in the absence of levamisole (data not shown). Altogether, these data demonstrate that mitochondria fragment with age as revealed by a decrease in mitochondrial length, an increase in mitochondrial circularity and an overall increasingly ‘fragmented’ morphology. Furthermore, they demonstrate that mitochondria lose volume with age as revealed by a decrease in mitochondrial area in the imaged planes.We also quantified mitochondrial length, circularity and area . We observed a decrease in mitochondrial length, an increase in mitochondrial circularity, and a decrease in mitochondrial area with age Figures . We obseC. elegans exhibit a shorter lifespan at 25°C in comparison to 15°C [Next, we asked if there is a correlation between the changes in mitochondrial morphology and aging.  to 15°C . We therWe observed a similar trend in case of mitochondrial circularity Figure . At Day In the case of mitochondrial area, we only observed a significant difference at Day 11 between animals grown at 15°C compared to 25°C and animals grown at 20°C compared to 25°C Figure . The quaage-1 (age-1(hx546)). age-1 encodes the C. elegans ortholog of the phosphoinositide 3-kinase (PI3K) and the mutation hx546 causes an extension of lifespan by ~40% [age-1(hx546) animals live longer than wild-type animals (age-1(hx546) animals  animals. The difference in mitochondrial length between the two strains was significant at Day 5 and at Day 11 . Most significantly, in age-1(hx546) animals, fragmented mitochondrial morphology was only observed at Day 11 whereas it was already observed at Day 1 in wild type  animals.To determine the effects of mutations in genes implicated in the aging process on the mitochondrial morphology observed, we repeated the analyses with animals of different genetic backgrounds. We first analyzed animals carrying a loss-of-function mutation in the gene  by ~40% , 35. We  animals . We obse1 Figure . In case1 Figure . The ana1 Figure . The quae Figure . In concdaf-16 encodes a Fork-head transcription factor and a loss-of-function mutation in daf-16 causes animals to live ~20% shorter [daf-16(mu86) animals live shorter than wild-type animals (daf-16(mu86) animals than in wild-type animals. For example, a significant difference in mitochondrial length was observed between wild type and daf-16(mu86) at Day 5  animals, an analysis at Day 16 was not possible. An analysis of mitochondrial circularity showed a significant difference between the two strains at Day 1 and at Day 5  animals, very fragmented mitochondrial morphology was already observed at Day 5, whereas in wild type, it was only observed at Day 11  animals. shorter , 13. We  animals . We obse animals . However5 Figure . Due to 5 Figure . In addi5 Figure . These o1 Figure . Hence, clk-1(e2519) mutant animals. clk-1 encodes a hydroxylase involved in the biosynthesis of the redox-active lipid ubiquinone (co-enzyme Q) and the clk-1 loss-of-function mutation e2591 causes the animals to live ~25% longer than wild-type [clk-1(e2591) animals live longer than wild-type animals (age-1(hx546) animals, changes in mitochondrial morphology and volume were delayed inclk-1(e2519) animals compared to wild-type animals  animals throughout the course of the experiment (Day 1 through Day 16)  Figure . In conc2 coefficient=0.1)  at Day 5 or Day 7 tend to have a longer lifespan, the overall correlation is low (R) Figure . In the ) Figure . In concC. elegans body wall muscle cells has previously been shown to change during aging, with mitochondria becoming progressively more swollen [C. elegans muscle cells fragment with age. Furthermore, using mitochondrial area as a measure of mitochondrial volume, we found that the volume of mitochondria in muscle cells is significantly reduced in old animals when compared to young animals. Such a decrease in mitochondrial volume is expected to lead to reduced mitochondrial activity, as evidenced by the finding that increasing mitochondrial volume can lead to increased mitochondrial activity [C. elegans as well as other organisms [The mitochondrial ultrastructure in  swollen . We pres swollen , mitochoactivity . The levactivity . For insactivity . Hence, rganisms -24, 26.clk-1 animals, at least in hypodermal cells, display a faster decrease in mitochondrial membrane potential with age than compared to wild type animals [Although a decrease in mitochondrial activity with age could explain the progressive loss in muscle function leading to sarcopenia, it has not been previously determined whether this will have a direct effect on lifespan. To address this question, we analyzed the kinetics of changes in mitochondrial morphology in different aging mutants. Interestingly, we discovered that mitochondrial fragmentation and the decrease in mitochondrial volume are accelerated in short-lived mutants and delayed in long-lived mutants. The extent of these changes therefore seems to correlate with the biological age of the animal. However, we found that the values of these parameters in a young adult animal cannot be used to reliably predict its lifespan. Therefore, our results suggest that mitochondrial morphology and volume cannot be used as bio-markers of aging. Although mitochondrial fragmentation likely causes an age-dependent decrease in mitochondrial activity, this decrease is unlikely to be the primary cause of aging as evidenced by the observation that long-lived  animals .C. elegans, mitochondrial fission is controlled by the dynamin-related GTPase DRP-1 [drp-1 loss-of-function mutation tm1108 have a normal lifespan, the inactivation of drp-1 by RNAi has been shown to decrease lifespan [Mitochondrial morphology is the result of a balance between mitochondrial fusion and fission. In se DRP-1 . Althougse DRP-1 , 43. Whelifespan , 43. Desage-1(hx546) animals [C. elegans animals, for example by raising them in liquid medium, in which C. elegans animals are thought to be more active. Conversely, one could test whether reducing C. elegans locomotion (by using mutants or drugs) will accelerate the fragmentation of the mitochondrial network and possibly decrease lifespan.Why mitochondria fragment with age remains unclear. It could be attributed to the increased oxidative stress observed in aging animals , as oxid animals . Additio animals . To testFinally, the mechanism or mechanisms through which mitochondrial volume decreases with age remain to be determined; however, we speculate that a decrease in mitochondrial volume could be caused by a reduction in mitochondrial biogenesis . AlternaC. elegans, it has been shown that the major source of lifespan variability is different pathogenicity from individual to individual [C. elegans, the production of cross progeny production through Day 4 is an early-stage biomarker of longevity [mir-71::GFP, mir-246::GFP and mir-239::GFP) can also be used to predict lifespan in C. elegans [The increased understanding of the control of longevity has generated considerable interest in the identification of biomarkers of aging. Studies using various model organisms and human tissue samples have led to the identification of a few biomarkers of aging that can predict lifespan with some success. For example, the state of DNA methylation in various tissues in humans and the length of telomeres in zebra-finches can be used as biomarkers of aging , 48. In dividual . Furtherongevity . Interes elegans . In our In summary, we show that mitochondrial fragmentation and a decrease in mitochondrial area correlate with age and are likely to participate in the degeneration of body wall muscles. We also present evidences that these changes are unlikely to be the primary cause of aging. Future studies are warranted to understand how these mitochondrial changes are induced during aging with the aim of increasing our understanding of sarcopenia and other age-related conditions.C. elegans strains were maintained and cultured as described previously [daf-16(mu86)[age-1(hx546)[clk-1(e2519) [bcIs78 is an integration of the Pmyo-3mitoGFP reporter allowing visualization of mitochondria in body wall muscle cells [bcIs78 males were crossed with hermaphrodites of the different mutant strains used in the study. F2 animals homozygous for both, bcIs78 and the respective mutation as well as F2 animals homozygous for bcIs78 only (wild-type control) were selected for studies with aging mutants.eviously . Mutatio-16(mu86) LG II, a-1(hx546), LG III,1(e2519) . Bristolle cells . To ensut-test.Mitochondrial length and circularity in body wall muscle cells were measured as described previously . MitochoThe same image datasets were used for analysis of mitochondrial area in body wall muscle cells. A 300 × 300 pixels region was selected from each image and mitochondrial area was determined by measuring the fluorescent area of the pixel region (pixel area) using Metamorph software. The data analysis was done double blind and average of both datasets was used for comparison. At least 9 images were analyzed for each time point per genotype.tubular; (2) images containing a combination of interconnected mitochondrial networks along with some smaller fragmented mitochondria were classified as intermediate; (3) images with a majority of short mitochondria were classified as fragmented; and finally, (4) images with sparse small round mitochondria were classified as very fragmented, as shown in Figure Qualitative assessment of mitochondrial morphology in body wall muscle cells was performed on the same image datasets using the whole image. Morphological categories were defined as follows: (1) images containing a majority of long interconnected mitochondrial networks were classified as Animals were grown at 20°C unless otherwise stated on NGM plates without using FUDR (5-fluorodeoxyuridine) and picked to fresh NGM plates after the L4 stage. The animals were subsequently picked to fresh NGM plates every two days until parched. Animals were scored by gentle prodding with a platinum wire. Injured, missing or bagged animals were censored. Graphpad Prism Software was used for Kaplan-Meier survival curve and log-rank test.bcIs78 animals were grown at 20°C on NGM plates without using FUDR (5-fluorodeoxyuridine ) and imaged at Day 5 and Day 7. The imaged animals were recovered from the agar pad and transferred onto individual NGM plates. Animals were scored by gentle prodding with a platinum wire. Animals that were injured during the transfer after imaging were not used for the analysis. The resulting data were visualized and the goodness of fit of linear regression was analyzed using Graphpad Prism Software.Imaging was performed at 20°C using a microscope equipped with a 100× 1.3 NA oil lens  and a charge-coupled device camera . MetaMorph software  was used to acquire fluorescent z stacks of individual animals (0.5 μm/slice). The acquisition time was 100 ms. Worms were immobilized during imaging using 25mM levamisole. Deconvolution was performed blind on the fluorescent z stack (10 iterations) using AutoDeblur/AutoVisualize software . Images shown in the figures correspond to single plane images.


Interleukin-10 (IL-10) is considered to be a cytokine with potent anti-inflammatory properties, which have been previously linked to increased incidence of sepsis. The level of IL-10 is elevated by cardiac surgery when cardiopulmonary bypass (CPB) and methylprednisolone are used. In our study, we compare the level of IL-10, IL-10 Receptor (IL-10R), and percentage of neutrophils between two groups of cardiac surgical patients undergoing Coronary Artery Bypass Grafting, both of which were not given methylprednisolone. The first group was operated with conventional CPB, while the second group was operated with minimally invasive CPB (mini-CPB). We detected enhanced level of IL-10 during surgery and at the end of surgery in both groups of patients. While no correlation between IL-10 and IL10R was found, IL-10 was positively correlated with increased percentage of neutrophils at the time points when the level of IL-10 peaked. Cardiac surgery is connected to profound inflammatory response, characterized by increased level of both proinflammatory and anti-inflammatory mediators. Cardiopulmonary bypass (CPB) is considered to be a potential trigger of cytokine release, therefore, the impact of cardiac surgery on morbidity and mortality is often correlated with the use of CPB , 2. SurgAs the blood reaches all the body compartments, inflammatory response to surgical injury exceeds local reaction and becomes systemic. Although the increased production of cytokines is essential for protection against infection and also healing the wounds, unregulated inflammatory response is likely to have a harmful effect. Regulating mechanisms consist of anti-inflammatory molecules such as cytokines, cell surface enzymes, receptor antagonists, and soluble receptors . The netα, is inhibited [IL-10 is a regulatory and immunosuppressive cytokine that is produced by a variety of cells . Upon binhibited .α have already been extensively evaluated in patients undergoing CABG surgery with conventional CPB [The changes in production of IL-10, IL-1, and TNF-n = 22) or mini-CPB (n = 22). Exclusion criteria consisted of acute inflammation, urgent operation, reoperation, combined operations, operative risk more than 5% (according to logistic Euroscore), preoperative level of serum creatinine above 130 μmol/L, hepatic disease, and malignancies. The demographic and preoperative data of our patients are shown in Forty-four patients, undergoing elective coronary artery bypass grafting (CABG) surgery on an arrested heart using CPB, were enrolled into our study. All patients were well informed about the purpose of the study and they confirmed their unconstrained participation by a written consent. The study project was approved by the Ethics Committee of the University Hospital in Hradec Kralove, Czech Republic. Patients, included in the study group in the period from December 2006 to December 2007, were randomly assigned to surgery either with the use of conventional CPB  were integrated into the extracorporeal circuit. The system surface was not treated with any hemocompatible substance. The priming solution consisted of 500 mL of Ringer's lactate, 500 mL of Rheodextran (Rheomacrodex), 5,000 IU heparin, 80 mL of Natrium Bicarbonate , 20 mL of 10% Magnesium Sulphate, and Mannitol (at 1 g/kg body weight). The priming volume was calculated so that hematocrit level reached above 0.22. Heparin was administered intravenously at 300 IU/kg body weight to maintain an activated clotting time (ACT) above 480 s during bypass procedure. Patients received neither aprotinin nor corticosteroids intravenously. Pump flow rates averaged 2.4 L/min/m2 of body surface area with pressure maintained at 50–60 mmHg. The patients were kept normothermic. Cardioplegic arrest was induced with a cold blood cardioplegic solution, which consisted of blood mixed with St. Thomas solution  in 4 : 1 ratio. It was administered antegradely into the aortic root with doses added every 20 min or as needed. All patients received an internal artery mammary graft to the left anterior descending coronary artery. The central aortovenous anastomoses were performed during the reperfusion phase of cardiopulmonary bypass with the heart beating. After the termination of CPB, heparin anticoagulation was antagonized using Protamine Sulphate at a dose of 1 : 1.CPB was established using a two-stage venous drainage and ascending aortic return. A roller pump , a membrane oxygenator  in a closed modification with a collapsible reservoir, a cardiotomy suction device, and a 40 μm arterial line filter, and a venous bubble trap. Cardiotomy suction was not used. The whole system, consisting of a closed loop with the surface treated with PH.I.S.I.O phosphorylcholine coating  and very short tubing, was placed close to the operating field. The priming solution, heparinization, pump flow, temperature, and surgery technique were identical with the conventional CPB procedure described above. Cardioplegic arrest, induced according to the Calafiore warm blood-cardioplegia protocol, was administered antegradely into the aortic root. At the beginning of CPB, crystalloid priming solution was flushed retrogradely together with the blood coming from the arterial line to minimize the hemodilution of the patient.Mini-CPB was established using a small 22F two-stage venous drainage and ascending aortic return. Minisystem Synergy  consisted of a centrifugal pump, membrane oxygenator, 40 2 0.5 was employed. Mean arterial pressure was kept above 50 mmHg, with norepinephrine administered as required.All patients were anesthetized according to the current protocol of our department. Anesthesia was induced using Thiopental and Midazolam. Muscular relaxation was achieved with Cisatracurium. Anesthesia was maintained with Isoflurane and intermittent use of Sufentanyl. Continuous infusion of Propofol was used as a supplement if needed. Volume-controlled ventilation with FiOBlood samples were withdrawn from subclavian vein before and during surgery and from antebrachial vein in postsurgery period. The samples were collected into anticoagulant-untreated Vacutainer tubes as well as heparinized Vacutainer tubes  at the following time points: before surgery (introduction of anesthesia), at the beginning of CPB, at termination of CPB, at the end of surgery, and on the 1st, 3rd, and 7th postoperative day.Serum was separated from the blood cells by centrifugation at 1000 g for 15 min. Samples were stored at −80°C prior to analysis. IL-10 concentration was determined by enzyme-linked immunosorbent assay (ELISA), with a sensitivity of 1 pg/mL. ELISA kit was purchased from Bender MedSystems (Austria). IL-10 concentrations were measured using spectrophotometer  with Genesis software.μL of blood was incubated with titrated monoclonal antibodies anti-CD3 FITC/IL-10R PE/CD14 PerCP/CD16 APC. Antibodies, except for anti-IL-10R, were purchased from BD Biosciences (USA), while anti-IL-10R was purchased from BioLegend, USA. Following the 20 min incubation, red blood cells were lysed by hypotonic lysis and then the samples were washed. The data acquisition was performed with FACS Calibur flow cytometer using CellQuest software , and the analysis was done using FlowJo software . Expression of IL-10R on cells was displayed as median fluorescence intensity (MFI). Expression of IL-10R during surgery was not assessed.Heparinized blood samples were processed immediately after collection. 50 U test. The relationship between IL-10 and percentage of neutrophils was assessed by Spearman correlation coefficient. Demographic and clinical data were analyzed by Fisher exact test, Mann-Whitney U test, and Student's t-test. All tests were performed at a significance level of 0.05. Bonferroni correction was used in case of multiple comparisons. Results of statistical analysis were expressed as medians unless stated otherwise.Values of IL-10, IL-10R, and percentage of neutrophils during and after surgery were compared to preoperative values. Normal distribution of the data was determined by Shapiro-Wilk test. Changes within a group were evaluated using ANOVA for repeated measures and Dunnett's test or Friedman ANOVA and Wilcoxon paired test. To determine the differences between both groups of patients, values of IL-10, IL-10R, and percentage of neutrophils were compared at matching time points using two-way ANOVA for repeated measures and Fisher's LSD test or Mann-Whitney P < 0.001), at the end of surgery , and on the 1st postoperative day . Similarly, the mini-CPB group experienced an increase in serum level at the termination of CPB , at the end of surgery , and on the 1st postoperative day . Postsurgery monitoring revealed that the level of IL-10 was gradually decreasing in both groups and did not significantly differ from preoperative levels  between both groups at the termination of CPB: the conventional CPB group topped the mini-CPB group by reaching the value of 52.7 pg/mL. At this time point, IL-10 in mini-CPB group was enhanced, but only reached 10.6 pg/mL. In mini-CPB group, the level of IL-10 was highest at the end of surgery (21.2 pg/mL). In postsurgery period, the level of IL-10 decreased and did not differ between both groups (There was significant serum-level difference (h groups .P < 0.001) in both groups of patients at the end of CPB, at the end of surgery, and also on the 1st and 3rd postoperative day. The baseline was 55% of neutrophils in conventional CPB group and 53% in mini-CPB group. The highest percentage of neutrophils was measured on the 1st postoperative day in both groups of patients . There was statistically significant difference in percentage of neutrophils between both groups at the end of surgery (P < 0.05) when conventional CPB group reached 79% and mini-CPB group reached 75% of neutrophils , at the end of surgery in conventional CPB group , and at the end of surgery in mini-CPB group   in conventional CPB group, as well as in mini-CPB, where the preoperative level dropped from MFI of 9.8 to 7.6 (P < 0.05). On the 3rd postoperative day, the expression of IL-10R on monocytes was significantly enhanced (P < 0.05) and 11.3 in mini-CPB (P < 0.05). After that, the IL-10R expression decreased again and there was no significant difference between the preoperative value and the value on the 7th day after surgery in both groups   . While wμmol/L [In previous works, IL-10 level was also found to be significantly increased in patients who suffered from postoperative renal dysfunction , which wμmol/L . The minrs = 0.73, P < 0.001) and at the end of surgery in mini-CPB group . We also found lower—but still significant—correlation  between IL-10 and percentage of neutrophils at the second highest level of IL-10 (at the end of surgery) in conventional CPB group. Our data suggests that neutrophils were the main producers of IL-10 in most of our cardiac surgical patients. However, the lower correlation coefficient at the end of surgery indicates other cells might also have participated in IL-10 production. The observation is in agreement with recently published findings that show that neutrophils are an important source of IL-10 [We compared the percentage of neutrophils to IL-10 level when IL-10 in serum was at its highest level. We observed significant correlation between IL-10 and percentage of neutrophils at the termination of CPB in conventional CPB group (of IL-10  and thatIL-10 exerts its function through the binding to IL-10R . It seemIL-10 level and percentage of neutrophils are significantly affected by the type of cardiac surgery employed. Although IL-10 level may have statistical relation to sepsis or renal dysfunction, the generally accepted critical level that would enable to unambiguously distinguish between patients with worse or good prognosis does not exist. However, in certain conditions, like using conventional CPB, IL-10 may represent a supporting tool, that, along with other parameters, would help rank the patients in likelihood of organ dysfunction or failure. The observed correlation between increased level of IL-10 and higher percentage of neutrophils in both groups of patients suggests functional relationship between both parameters.


P) < 0.0001). However, there appeared high correlation between them in view of statistics (R-square (R) > 0.8). It is shown that one needs to decide SUVs-maximum for PET/MRI with the reduction of 25.0~26.4% from their evaluated value and needs to decide with the reduction of 28.8~29.4% in the same situation but with the use of contrast media. The use of SUVLBM-maximum (SUVLean Body Mass-maximum) is very advantageous in reading overall image of PET/CT and PET/MRI to medical doctors and researchers, if we consider its convenience and efficiency. We expect that this research enhances the level of the early stage accurate diagnosis with whole-body images of PET/MRI and PET/CT.The purpose of this research is to establish an overall data set associated with the VOI (Volume of Interest), which is available for simultaneous assessment of PET/MRI and PET/CT regardless of the use of contrast media. The participants as objects of this investigation are 26 healthy examinees in Korea, SUV s-maximum evaluation for whole-body F-18 FDG (fluorodeoxyglucose) PET/MRI image using VOI of normal region has exhibited very significant difference to that for whole-body F-18 FDG PET/CT image (significant probability value ( The introduction of recently developed imaging apparatus PET/MRI that combines PET (positron emission tomography) and MRI (magnetic resonance imaging) gives very promising anticipation as a step for new and more powerful acquisition of body images . The widThe merit of the one-unit PET/CT is that not only can we establish CT data using X-ray but also it enables us to achieve attenuation correction (AC) for PET image using CT value  of CT image . HoweverStandardized uptake values (SUVs) are an important index for evaluating the accuracy of cancer diagnosis carried out via inspection of F-18 FDG (fluorodeoxyglucose) PET/CT and the effects of their cure. Hence, if we consider that F-18 FDG sticks fast to some parts of the internal organs physiologically, the pathological data can be estimated from SUVs. For the case of the intake of F-18 FDG, the diversity of SUVs is essential in order to understand normal organization of the human body .Many researchers have evaluated the equation of SUVs associated with biological or physical factors after setting up the size of the region of interest (ROI) . BecauseLBM-maximum using ROI relevant to 2-dimensional area in PET/CT.Recently, many researchers take body weight (BW) considering the weight of patient, body surface area (BSA) considering the weight without fat, and lean body mass (LBM) considering the area of the surface of body as reference factors for the results they obtained , 11 and In this work, we intend to enhance the level of early/precise diagnosis for positive regions such as cancers and inflammations via the research of SUVs-maximum using 3-dimensional VOI, which is recently enabled with the introduction of PET/MRI equipment in hospitals. In particular, we try to establish the data sets which enable us to simultaneously evaluate images of PET/CT and PET/MRI regardless of the use of contrast media. The final purpose of this research is to compare and analyze SUVs-maximum statistically for whole-body image obtained from PET/CT and PET/MRI for healthy examinees.The number of patients who became the object of our survey is 26. They had taken PET/MRI examination immediately after the examination of whole-body FDG PET/CT from July 2012, to August 2012. This research is carried out under the permission of Institutional Review Board (IRB) of Korea. They are six men and twenty women. Their age is distributed from 35 to 75 and the average age is 52.5. Distributed stature is from 145 cm to 177 cm and their average is 160.0 cm. The range of weight is from 40 kg to 76 kg and their average weight is 57.8 kg. The number of examinees who take contrast media is 16 and who do not take is 10.The examinees fasted at least six hours before they take examination and F-18 FDG is injected into the necessary parts of their body after meeting a required condition that their checked blood-glucose level is lower than 180 mg/dL. F-18 FDG 8.14 MBq/kg is injected in the examinees' vein and, after 60~90 minutes, the PET/CT  images are acquired at first. The AC for the obtained CT image  is fulfilled and then we obtained 7~9 beds of 3D-mode emission images in the same range as the primary CT scan. The time taken for each scan is about 3 minutes. The PET image is reconstructed to be OSEM (ordered-subsets expectation maximization method) algorithm mode, and AC is carried out using CT image.The examinees who had taken PET/CT examination had been immediately examined PET/MRI  in order to get the required images. After the first image from head to proximal femur is obtained (it takes about 19 seconds), 4 beds of 3D mode emission image are obtained in the same range as the CT scan, where the acquirement of each bed takes two minutes. PET images are reconstructed to be the OSEM algorithm mode and AC is carried out using Dixon VIBE MRI images.1 weight MRI pulse sequence based on 2-point Dixon 3D volume is applied under the condition of inducting regular breathing and, after that, the scanning table is moved to scan along head/neck, breast, abdomen, and pelvis in turn.Four body coils are set up in front of head coil in order to optimize signal-to-noise ratio (SNR) and spine clusters are equipped in the rear of it. Coronal plane T3 (in-plane resolution × slice thickness), acquisition time 18 s, repetition time (TR)/echo time (TE) 3.6/1.225, matrix 79 × 192, number of excitations 1, FOV 500 mm, phase FOV 72%, 1 slab with 128 slices, slice thickness 2.6 mm, flip angle 10°, and bandwidth 960 kHz.The variables relevant to obtaining MRI image are as follows: integrated parallel acquisition technique factor 2, voxel size 4.1 × 2.6 × 3.1 mmk-space acquisition in the middle is selected. Contrast media are not used in the inspection taken with MRI and raw images that automatically generate T1 weight in-/out-of-phase, water-only image, and fat-only image are used.To minimize artifacts produced through irregular breathing, PET data has been attenuation corrected and they are reconstructed as a mode of OSEM data. Attenuation maps are obtained from CT data produced by two lines in the existing mode which uses postprocess software tool of PET/CT scanner.Since inaccurate registration induces a significant problem in determining proper quantity of SUVs, attenuation correction factors are chosen delicately in this research and they are 0.1/cm for background, 0.018/cm for lung, 0.086/cm for fat, and 0.1/cm for soft tissue. This AC is used for reconstructing PET image on the basis of regularization of simulated MRI-based AC (PETAC_MRI) for overall items such as in the case of PETAC_CT.Raw data, CT data, PETAC_CT, and PETAC_MRI associated with the four parts of image  produced on the basis of Dixon MRI pulse sequence are transferred to workstation . Although all images enrolled in syngo.via are automatically evaluated, they can also be locally adjusted by hand if necessary. In PETAC_MRI, all uptaken portions are exactly represented in the merged anatomic image. Coronal plane, sagittal plane, and transverse plane are represented in monitor using 3D Fusion MM Oncology in order to evaluate the ability of quantity representation of PET/CT and PET/MRI. We referred  for anat3), spleen , aorta , bone marrow , lumbar 5 , and cerebellum  are selected and drawn. The errors of the range of measurements are confined within ±0.01% in this drawing. We also fitted anatomical positions of volume in coronal plane, sagittal plane, and transverse plane using the two lines  designed for Microsoft Windows operating system in managing all statistical processes with measured data. Linear regression analysis for SUVs-maximum of PET/CT and PET/MRI is performed and corresponding comparison of each normal area is carried out according to corresponding sample LBM-maximum for patients taking both PET/CT and PET/MRI shows very significant correlation between PET/CT SUVLBM-maximum and PET/MRI SUVLBM-maximum, which is 0.873 (P < 0.01). Equation of regression derived from this analysis is as follows:The results of linear regression analysis performed via computing variation of SUVB is that PET/MRI SUVLBM-maximum increases to the range 0.48–1.46 when PET/CT SUVLBM-maximum grows 1 unit . The regression equation derived from this analysis isThe linear regression analysis performed via variation calculation of SUVB is that PET/MRI SUVBW-maximum increases the amount of the range 0.770–1.672 as the PET/CT SUVBW-maximum grows 1 unit . The regression equation derived from this analysis is as follows:The results of linear regression analysis obtained from variational calculation of SUVB is that PET/MRI SUVBSA-maximum increases the amount of the range 0.011–1.535 as the PET/CT SUVBSA-maximum grows 1 unit . The regression equation obtained from this analysis can be written asThe results of linear regression analysis obtained from the variational calculation for the case of the patients taking both PET/CT and PET/MRI reveal that the correlation between PET/CT SUVBW-maximum increases the amount of the range 0.225–0.790 when PET/CT SUVBW-maximum grows 1 unit . The equation of regression derived from this analysis is The meaning of 95% confidence interval for regression coefficient is that PET/MRI SUVBSA-maximum increases the amount of the range 0.569–1.048 as the PET/CT SUVBSA-maximum grows 1 unit Y=−0LBM-maximum in PET/MRI image decreased significantly with the reference PET/CT image for the eight normal areas taken without using contrast media (P < 0.05). It turned out that PET/MRI exhibits a total of 26.3% difference with the reference to PET/CT: spleen showed the largest difference (32.1%) whereas aorta the smallest difference (18.2%). All of SUVLBM-maximum is significantly decreased with the reference to that of the PET/CT image of the eight normal areas taken with the use of contrast media (P < 0.01).All of SUVA total of 29.3% difference appeared in PET/MRI with reference to PET/CT, the highest difference is appeared in spleen (35.8%), and the lowest difference is in aorta (22.8%). It turned out that the case using contrast media exhibits 3.0% higher difference than that without using contrast media .BW-maximum in PET/MRI image appeared to decrease significantly with reference the eight areas of PET/CT image without using contrast media (P < 0.05). There appeared 25.0% difference in PET/MRI image with reference to PET/CT image and the largest difference is shown in lumbar 5 (33.6%) and the smallest difference in aorta (15.7%). All of SUVBW-maximum in PET/MRI image decreased significantly with reference to eight normal areas PET/CT image obtained with the use of contrast media (P < 0.01). PET/MRI image showed a total of 28.8% difference from that in PET/CT image: the largest difference appeared in liver (35.8%) whereas the smallest in aorta (17.6%). The difference of the case using contrast media is higher by 3.7% than that without using contrast media . SUVBSA-maximum for PET/MRI showed a total of 26.4% difference from that for PET/CT: the largest difference appeared in lumbar 5 (33.3%) whereas the smallest in aorta (18.0%). All of SUVBSA-maximum in PET/MRI image are decreased significantly compared to those of the eight normal areas of PET/CT image obtained with the use of contrast media (P < 0.01). PET/MRI image shows a total of 29.4% difference from that of PET/CT image: the largest difference appeared in spleen (36.2%) whereas the smallest difference is in aorta (18%). The case using contrast media exhibits 2.9% higher difference than that without using contrast media  is more or less degraded when it is compared to that of CT (512 × 512 matrix and a soft tissue kernel). However, the research of Martinez-Möller shows that the enhancement of contrast for soft tissue [There is a trend that space resolution of PET/MRI  [BW-mean for normal area is 0.945, 95% with confidence interval 0.273~0.922 (P < 0.0001). From this difference, we conclude that cancer tissue accumulates more FDG than normal area.The research of Martinez-Möller shows that correlation coefficients of SUV 0.0001) . The resAnother difference in scintillator is that fluorescent materials used in PET/CT and PET/MRI are made in the same company and that is lutetium oxyorthosilicate fluorescent material. Concerning this research, LSO fluorescent material is used for PET/MRI and BGO (bismuth germanium oxide); fluorescent material is used for PET/CT, leading us to conjecture that there is a difference of sensitivity between them.LBM-maximum and SUVBW-maximum. In general statistics, we see that correlation coefficients higher than 0.8 are regarded as high correlation, between 0.6~0.8 are normal, 0.4~0.6 are low, and below 0.4 are evaluated as uncorrelated. Hence, when we evaluate image regardless of using contrast media, the use of SUVLBM-maximum is recommended. In fact, SUVLBM-maximum is also used in previous actual researches since the average value of standardized uptake values is unstable every time when we acquire them due to irregular size of positive regions [In this research, the cases in which correlation coefficient is higher than 0.8 are SUV regions .SUVs are affected by various factors which are enumerated as follows. At first, there is a method in which physical constitution of patient is evaluated relatively by putting the specific gravity of body as 1. The index of blood glucose is a factor that disturbs the absorption of FDG in cancer. In case of PET, since the positive region reveals higher absorption rate than their environment, if the size of the measuring object is not sufficiently larger than the space resolution (at least more than 5 times), the measured radioactivity is lower than the actual one.The data of SUVs-maximum measured from the normal eight positions are uniform for the case of F-18 FDG. However, in general, there is a limit when diagnosing cancer and inflammation in connection with high absorption rate because it is difficult to obtain physiological upper threshold. Hence it is required to know SUVs-maximum of normal area in PET/CT and PET/MRI as an appropriate counterplan to reduce false positivity and false negativity .LBM-maximum when we read overall image of PET/CT and PET/MRI. We expect future-oriented active research for PET/MRI image in the upcoming studies.Considering conveniency and efficiency for medical doctors and researchers, it is useful to use SUVLBM-maximum evaluation using VOI of normal area for whole-body F-18 FDG PET/CT and whole-body F-18 FDG PET/MRI exhibits very significant difference with reference to PET/CT (P < 0.0001). In addition to this, they exhibit high correlation statistically (R > 0.8) and SUVLBM-measured shows the lowest rate of reduction among various data managed regardless of using contrast media. It is necessary to judge with reduction of 25.0~26.4% when we evaluate SUVs of PET/MRI, whereas we should reduce 28.8~29.4% when we judge it with the use of contrast media.SUV